1. The Constant Need for New Compounds and Biologically Active Compounds for the Development of New Pharmaceutics and Medicaments
The advance in medical research constantly leads to the discovery of yet unknown and complex diseases, for which new, specific and effective pharmaceuticals and treatments have to be developed. In a majority of such new cases, nothing is known about biological compounds which would/could be effective in treating such diseases.
In general, time plays an important role in these cases, since in most of the cases an effective drug/treatment has to be found very rapidly.
Furthermore, such developments currently involve very cost-intensive screening procedures until a particularly suited compound (often called “lead”-compound) is found which could then serve as a chemical basis for an effective treatment.
Another current development in the treatment of diseases is the so-called “personalized” treatment, in which an individually treatment schedule and/or pharmaceutical composition is applied to the individual patient. Since the treatment is directed or applied to a very limited scope and number of patients (i.e. only one patient) and diseases, such treatment again is very cost-intensive and therefore can only seldom be applied in an efficient manner.
Furthermore, problems arise with already known biological compounds in such a way that a) unwanted side effects are discovered, that limit the use of established pharmaceutics, and b) resistance can be found/are developed against major therapeutics (like in the case of antibiotic resistances) which limit the success of presently applied compounds.
In view of the above, there exists a constant need for new potential candidate compounds for the treatment of emerging new diseases, personalised medicine and, of course, alternative treatments for already known diseases. Furthermore, the need exists for a reliable, cost-effective, fast and automateable method for screening such new effective compounds.
2. Screening for New Biologically Active Compounds Using “Combinatorial Chemistry”
The method of combinatorial chemistry is described as a profound change in the strategies that biotechnology-based industries are deploying in the search for exploitable biology and to discover new products and develop new or improved processes. (see, for example, Bull A T, et al. “Search and discovery strategies for biotechnology: the paradigm shift.” Microbiol Mol Biol Rev 2000 September; 64(3):573-606)
In general, combinatorial chemistry involves screening of a specific (or a set of specific) compound with a vast number of potential biological candidate substances (for example, proteins) that might interact with the compound. Interacting partners are selected and used for further screening. Initially screened and isolated compounds can be used as “lead”-compounds for the development of biologically active compounds useful for treatment of diseases.
Other methods and devices for combinatorial chemistry are described in, for example, U.S. Pat. No. 6,175,816 (Flavin, et al.; “Use of automated technology in chemical process research and development”) U.S. Pat. No. 6,045,755 (Lebl, et al.; “Apparatus and method for combinatorial chemistry synthesis”) U.S. Pat. No. 5,880,972 (Horlbeck; “Method and apparatus for generating and representing combinatorial chemistry libraries”) and U.S. Pat. No. 5,721,099 (Still, et al.; “Complex combinatorial chemical libraries encoded with tags”).
WO 00/71742 describes the “marriage” of solid-state electronics and neuronal function to create a new high-throughput electrophysiological assay to determine a compound's acute and chronic effect on cellular function. Electronics, surface chemistry, biotechnology, and fundamental neuroscience are integrated to provide an assay where the reporter element is an array of electrically active cells. This innovative technology was applied to neurotoxicity, and to screening compounds from combinatorial chemistry, gene function analysis, and basic neuroscience applications. Further disclosed are algorithms to analyze the action potential peak shape differences to indicate the pathway(s) affected by the presence of a new drug or compound; from that, aspects of its function in that cell are deduced. This observation is said to be exploited to determine the functional category of biochemical action of an unknown compound.
WO 00/23458 describes templated combinatorial chemical libraries comprised of a plurality of bifunctional molecules having both a chemical compound and a nucleic acid tag that defines the structure of the chemical compound and directs its synthesis.
Logani S, et al. (“Actions of Ginkgo Biloba related to potential utility for the treatment of conditions involving cerebral hypoxia.” Life Sci 2000 Aug. 11; 67(12):1389-96) describe the use of HTS (high-throughput screening) libraries for reevaluation of the pharmacologic properties of substances such as extract from the leaves of Ginkgo biloba Linne (form. Salisburia adiantifolia Sm.).
Although the method of combinatorial chemistry exhibits several advantages in comparison to conventional methods for screening for biologically effective compounds which are useful for the development of new medicaments, there are still several drawbacks associated with this method.
The screening of a combinatorial chemistry library involves a screening for a multitude of different possible reactions and/or interactions of the compounds to be analysed with the interacting partners. Therefore, the reaction conditions are assumed crucial for the result of the screening. In particular, a compound which shows an interaction with a target in such a combinatorial assay in vitro might exhibit completely different reaction conditions in vivo which makes prediction of an effective compound very difficult and unreliable. As a result, an interaction in an in vitro combinatorial chemistry screening assay can always only give a hint for a potential biological function of the screened compound in vivo.
As a result, combinatorial chemistry screening involves a necessary second step; once a potential target/lead compound has been identified/found, the biological effect still has to be confirmed/determined in an in vivo context. This makes compound identification using this method unpredictable, slow and costly.
3. Methylation Pattern and Diseases
3.1 State of the Art in Methylation Analysis
The modification of the genomic base cytosine to 5′-methylcytosine represents the epigenetic parameter which to date is the most important one and has been best examined. Nevertheless, methods exist today to determine comprehensive genotypes of cells and individuals, but no comparable methods exist to date to generate and evaluate epigenotypic information on a large scale.
In principle, there are three methods that differ in principle for determining the 5-methyl state of a cytosine in the sequence context.
The first method is based in principle on the use of restriction endonucleases (RE), which are methylation-sensitive”. REs are characterized in that they produce a cut in the DNA at a certain DNA sequence which is usually 4-8 bases long. The position of such cuts can be detected by gel electrophoresis, transfer to a membrane and hybridization. Methylation-sensitive means that certain bases within the recognition sequence must be unmethylated for the step to occur. The band pattern after a restriction cut and gel electrophoresis thus changes depending on the methylation pattern of the DNA. However, most CpG that can be methylated are outside of the recognition sequences of REs, and thus cannot be examined.
The sensitivity of this method is extremely low (Bird, A. P., Southern, E. M., J. Mol. Biol. 118, 27-47). A variant combines PCR with this method; an amplification by two primers located on both sides of the recognition sequence occurs after a cut only if the recognition sequence is in the methylated form. In this case, the sensitivity theoretically increases to a single molecule of the target sequence; however, only individual positions can be examined, at great cost (Shemer, R. et al., PNAS 93, 6371-6376).
The second variant is based on the partial chemical cleavage of whole DNA, using the model of a Maxam-Gilbert sequencing reaction, ligation of adaptors to the ends thus generated, amplification with generic primers, and separation by gel electrophoresis. Using this method, defined regions having a size of less than thousands of base pairs can be examined. However, the method is so complicated and unreliable that it is practically no longer used (Ward, C, et al., J. Biol. Chem. 265, 3030-3033).
A new method for the examination of DNA to determine the presence of 5-methylcytosine is based on the specific reaction of bisulfite with cytosine. The latter is converted under appropriate conditions into uracil, which, as far as base pairing is concerned, is equivalent to thymidine, and which also corresponds to another base. 5-Methylcytosine is not modified. As a result, the original DNA is converted in such a manner that methylcytosine, which originally could not be distinguished from cytosine by its hybridisation behaviour, now can be detected by “normal” molecular biological techniques. All of these techniques are based on base pairing, which can now be completely exploited. The state of the art, as far as sensitivity is concerned, is defined by a method which includes the DNA to be examined in an agarose matrix, intended to prevent the diffusion and renaturing of the DNA (bisulfite reacts only with single-stranded DNA) and to replace all precipitation and purification steps by rapid dialysis (Olek, A., et al., Nucl. Acids. Res. 24, 5064-5066). Using this method, individual cells can be examined, which illustrates the potential of the method. However, so far only individual regions up to approximately 3000 base pairs in length have been examined, and an overall examination of cells to identify thousands of possible methylation events is not possible. However, this method is not capable of reliably analyzing minute fragments from small sample quantities. In spite of protection against diffusion, such samples are lost through the matrix.
3.2 State of the Art in the Use of the Bisulfite Technique
To date, barring few exceptions, (for example, Zeschnigk, M. et al., Eur. J. Hum. Gen. 5, 94-98; Kubota, T. et al., Nat. Genet. 16, 16-17), the bisulfite technique is only used in research. However, short specific pieces of a known gene after bisulfite treatment are routinely amplified and either completely sequenced (Olek, A. and Walter, J., Nat. Genet. 17, 275-276) or the presence of individual cytosine positions is detected by a “primer extension reaction” (Gonzalgo, M. L. and Jones, P. A., Nucl. Acids. Res. 25, 2529-2531), or enzyme cut (Xiong, Z. and Laird, P. W., Nucl. Acids. Res. 25, 2532-2534). All these references are from the year 1997. The concept of using complex methylation patterns for correlation with phenotypic data pertaining to complex genetic diseases, much less via an evaluation algorithm such as, for example, a neural network, has, so far, gone unmentioned in the literature; moreover, it cannot be performed according to the methodologies of the state of the art.
3.3 State of the Art with Respect to Methylation and the Diagnosis of Human Diseases
In the past, modification of the methylation pattern was analysed in order to study and understand the genetic mechanisms which are involved in the outbreak or the progression of a disease. All this research was done in a piece-by-piece fashion by studying only one gene/chromosomal region at a time and no diagnosis/therapeutic treatment regimen was based on the methylation pattern modifications. In fact, the type of disease associated with the modification of the methylation pattern was known before methylation analysis was performed. Therefore, the following publications only indicate the wide-spread connection between modifications of the methylation patterns and human diseases. Modifications can include both hyper- or hypomethylation of selected sites of the DNA.
Disease associated with a modification of the methylation patterns are, for example:                Leukemia (Aoki E et al. “Methylation status of the p15INK4B gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes” Leukemia 2000 April; 14(4):586-93; Nosaka K et al. “Increasing methylation of the CDKN2A gene is associated with the progression of adult T-cell leukemia” Cancer Res 2000 Feb. 15; 60(4):1043-8; Asimakopoulos F A et al. “ABL1 methylation is a distinct molecular event associated with clonal evolution of chronic myeloid leukemia” Blood 1999 Oct. 1; 94(7):2452-60; Fajkusova L. et al. “Detailed Mapping of Methylcytosine Positions at the CpG Island Surrounding the Pa Promoter at the bcr-abl Locus in CML Patients and in Two Cell Lines, K562 and BV173” Blood Cells Mol Dis 2000 June; 26(3):193-204; Litz C E et al. “Methylation status of the major break-point cluster region in Philadelphia chromosome negative leukemias” Leukemia 1992 January; 6(1):35-41)        Head and neck cancer (Sanchez-Cespedes M et al. “Gene promoter hypermethylation in tumors and serum of head and neck cancer patients” Cancer Res 2000 Feb. 15; 60(4):892-5)        Hodgkin's disease (Garcia J F et al. “Loss of p16 protein expression associated with methylation of the p16INK4A gene is a frequent finding in Hodgkin's disease” Lab Invest 1999 December; 79(12):1453-9)        Gastric cancer (Yanagisawa Y et al. “Methylation of the hMLH1 promoter in familial gastric cancer with microsatellite instability” Int J Cancer 2000 Jan. 1; 85(1):50-3)        Prostate cancer (Rennie P S et al. “Epigenetic mechanisms for progression of prostate cancer” Cancer Metastasis Rev 1998-99;17(4):401-9)        Renal cancer (Clifford S C et al. “Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene and allelic losses at chromosome arm 3p in primary renal cell carcinoma: evidence for a VHL-independent pathway in clear cell renal tumourigenesis” Genes Chromosomes Cancer 1998 July; 22(3):200-9)        Bladder cancer (Sardi I et al. “Molecular genetic alterations of c-myc oncogene in superficial and locally advanced bladder cancer” Eur Urol 1998;33(4):424-30)        Breast cancer (Mancini D N et al. “CpG methylation within the 5′ regulatory region of the BRCA1 gene is tumor specific and includes a putative CREB binding site” Oncogene 1998 Mar. 5; 16(9):1161-9; Zrihan-Licht S et al. “DNA methylation status of the MUC1 gene coding for a breast-cancer-associated protein” Int J Cancer 1995 Jul. 28; 62(3):245-51; Kass D H et al. “Examination of DNA methylation of chromosomal hot spots associated with breast cancer” Anticancer Res 1993 September-October; 13(5A):1245-51)        Burkitt's lymphoma (Tao Q et al. “Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma: molecular analysis of primary tumor tissue” Blood 1998 Feb. 15; 91(4):1373-81)        Wilms tumor (Kleymenova E V et al. “Identification of a tumor-specific methylation site in the Wilms tumor suppressor gene” Oncogene 1998 Feb. 12; 16(6):713-20)        Prader-Willi/Angelman syndrome (Zeschnigh et al. “Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method” Human Mol. Genetics (1997) (6)3 pp 387-395; Fang P et al. “The spectrum of mutations in UBE3A causing Angelman syndrome” Hum Mol Genet 1999 January; 8(1):129-35)        ICF syndrome (Tuck-Muller et al. “CMDNA hypomethylation and unusual chromosome instability in cell lines from ICF syndrome patients” Cytogenet Cell Genet 2000;89(1-2):121-8)        Dermatofibroma (Chen T C et al. “Dermatofibroma is a clonal proliferative disease” J Cutan Pathol 2000 January; 27(1):36-9)        Hypertension (Lee S D et al. “Monoclonal endothelial cell proliferation is present in primary but not secondary pulmonary hypertension” J Clin Invest 1998 Mar. 1; 101(5):927-34)        Pediatric Neurobiology (Campos-Castello J et al. “The phenomenon of genomic “imprinting” and its implications in clinical neuropediatrics” Rev Neurol 1999 Jan. 1-15; 28(1):69-73)        Autism (Klauck S M et al. “Molecular genetic analysis of the FMR-1 gene in a large collection of autistic patients” Hum Genet 1997 August; 100(2):224-9)        Ulcerative colitis (Gloria L et al. “DNA hypomethylation and proliferative activity are increased in the rectal mucosa of patients with long-standing ulcerative colitis” Cancer 1996 Dec. 1; 78(11):2300-6)        Fragile X syndrome (Hornstra I K et al. “High resolution methylation analysis of the FMR1 gene trinucleotide repeat region in fragile X syndrome” Hum Mol Genet 1993 October; 2(10):1659-65)        Huntington's disease (Ferluga J et al. “Possible organ and age-related epigenetic factors in Huntington's disease and colorectal carcinoma” Med Hypotheses 1989 May; 29(1):51-4)        
All the above-cited documents are hereby incorporated by reference.
Furthermore, it is known that the methylation pattern of methylation sensitive sites of other genes that are associated with other diseases is modified during the acute or non-acute phases of these diseases. Those genes are depicted in the listing of genes that is enclosed in this application and are associated with, for example, diseases related to angiogenesis, apoptosis, behavior, disorders of the cell cycle, cell signalling, developmental disorders, diseases related with DNA adducts, DNA damage, disorders in DNA replication, gene regulation, diseases related to immunological disorders, disturbances of the metabolism, metastasis, diseases related to miscellaneous clinical syndromes, pharmacological conditions, diseases related to a disturbed signal transduction, disturbed transcription, and tumour suppression/oncogene related diseases.